Field of the Invention
The present invention relates to a method for producing a multimeric protein, which includes a monomeric protein obtained by fusing a first protein having an immunoglobulin fold structure to a second protein that can serve as a subunit structure.
Brief Description of the Related Art
Therapeutic antibodies have been successful because of their high therapeutic effect and low risk of side effects, but now have a problem of high medical fees due to the high production cost. There are other problems such as the therapeutic effect of such antibodies is limited to a relatively narrow range of diseases such as leukemia and autoimmune diseases, and the antibodies are not so effective against solid cancers and the like in which the distribution of a drug to the tissues is limited.
After the production of antibody fragments in Escherichia coli became possible in 1988 (Science, 240, 1038-1041 (1988), Proceedings of the National Academy of Sciences U.S.A., 85, 5879-5883 (1988), and Science, 242, 423-426 (1988)), modified antibodies (antibody fragments) having a high tissue distribution property and a lower molecular weight were developed. Many antibody fusion proteins have been proposed, which are obtained by fusing an antibody fragment to an anticancer agent, toxin, radioisotope, prodrug activating enzyme, or the like to obtain a higher cancericidal effect.
However, although the bivalent binding of naturally occurring antibodies serves as a basis of demonstrating a high affinity for a target antigen and excellent biokinetics, the binding activity to a target antigen of artificially produced monovalent antibody fusion proteins is far from the binding activity of naturally occurring antibodies.
Hence, recently, efforts have been actively made to produce multivalent low-molecular-weight modified antibodies and antibody fusion proteins (Nature Reviews Immunology, 10 345-352 (2010)). If it is possible to produce multivalent molecules like antibodies using a microorganism at a low cost, both a high performance and a low medical fee could be achieved.
Methods are known for producing multivalent molecules by utilizing the association of an antibody structure itself like those of antibodies mimicking diabody, triabody, or an immunoglobulin M. Other methods include a method for obtaining multivalent molecules by fusing a fragment structure of an antibody to an associative protein. Examples of such fusion include one utilizing dimerization via a leucine zipper structure linked to a single-chain antibody fragment of variable region (scFv), or via another design (Biochemisry (Mosc), 31, 1579-1584 (1992)), one achieved through a human p53-derived tetrameric α-helix structure (Journal of Immunogy, 157, 2989-2997 (1996))), one utilizing tetramerization of microorganism-derived streptavidin, and the like. Particularly, regarding the method in which streptavidin is fused, improved performances (such as increased stability and solubility, and decreased immunogenicity) have been already achieved by modifying the structure of streptavidin (Journal of Controlled Release 65, 203-220 (2000)). A protein fused to streptavidin enables drug pre-targeting using a biotin molecule, which binds to streptavidin with a high affinity. Thus, the fusion protein is one of the most promising multivalent fusion proteins (Journal of Controlled Release 65, 203-220 (2000)).
A problem thereof is that when a multivalent fusion protein is produced using a microorganism such as Escherichia coli, insoluble proteins are formed in cells of the microorganism in many cases. The insoluble proteins in cells of the microorganism have lost the activity and/or stability. Accordingly, if such proteins are used as a pharmaceutical preparation or the like, refolding operations for reconstructing the active structure are additionally required.
Schultz et al. have reported an example where an active form which is soluble in cells of Escherichia coli was produced by contriving a peptide linker in the scFv to be bound to streptavidin and the arrangement order of the H and L chains (Cancer Research, 60, 6663-6669 (2000)). Nevertheless, it is known that if altered to a modified form, streptavidin becomes insoluble in cells of Escherichia coli (Protein Science 10, 491-503 (2001)). Hence, it seems that refolding operations are required for streptavidin fusion proteins to be practically used.
In view of such circumstances, Choe et al. studied an example where a modified streptavidin fusion protein was refolded (Bulletin of Korean Chemical Society, 30, 1101-1106 (2009)). However, the recovery rate of the target was far below 1%, and there are still many problems to be solved for the practical use.
The present inventors have previously proposed a method (refolding method) for restoring a higher-order structure of a native state of a protein (denatured protein) which has lost its activity and/or stability as having become insoluble or lost a higher-order structure thereof (International Patent Application Publication No. WO2009/136568). According to this method, the protein structure can be effectively reconstructed by solubilizing a denatured and precipitated protein at pH 6.5 to 9.0 using a 1 to 3% predetermined aqueous solution of an acyl glutamic acid surfactant, and then lowering the concentration of the surfactant in the solubilized solution down to 0.02 to 0.5% using an arginine buffer.